Atovaquone pharmaceutical compositions

ABSTRACT

The invention relates to a process for the production of microfluidized particles of atovaquone having improved bioavailability.

This is a rule 60 file wrapper continuation of application Ser. No.08/448,370, filed May 31, 1995, now abandoned which is a 371 ofPCT/GB93/02646 filed Dec. 23, 1993.

The present invention relates to microfluidised particles of2-[4-(4-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone and to amethod for preparing them. More particularly the invention is concernedwith a pharmaceutical composition containing microflulidised particlesof 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone("atovaquone") and its use in therapy.

Atovaquone has previously been disclosed, for example in European PatentNo. 0123238 and U.S. Pat. No. 50,53,432 (incorporated herein byreference) which relates to 2-substituted-3-hydroxy-1,4-naphthoquinonesof formula (I): ##STR1## wherein either R¹ is hydrogen and R² isselected from C₁₋₆ alkoxy, aralkoxy, C₁₋₆ alkyl-C alkoxy, phenylsubstituted by one or two groups selected from halogen and C₁₋₆ alkyl,halogen and perhalo-C₁₋₆ alkyl or R¹ and R² are both C₁₋₆ alkyl orphenyl, and n is zero or 1, and physiologically acceptable salts thereofThe compounds are said to have antiprotozoal activity. Specifically,compounds of formula (I) wherein n is zero are said to be active againstthe human malaria parasite Plasmodium falciparum and also againstEimeria species such as E.tenella and E.acervulina which are causativeorganisms of coccidiosis and compounds of formula (I) where n is 1 aresaid to be active against protozoa of the genus Theileria, in particularT.annulata or T.parva. Amongst the compounds specifically named andexemplified is the compound of formula (I) wherein n is zero, R¹ ishydrogen and R² is 4-chlorophenyl, i.e. atovaquone.

EP 0362996 discloses the use of atovaquone in the treatment and/orprophylaxis of Pneumocystis carinii pneumonia.

Further uses of atovaquone against Toxoplasmosis and Cryptosporidiosisare disclosed in European patent application nos. 0445141 and 0496729respectively.

The efficacy of atovaquone as a therapeutic agent is limited by itsbioavailability. Accordingly it is an object of the present invention toprovide atovaquone in a more bioavailable form.

It has now been found that the bioavailability of atovaquone can beincreased by ensuring that the particle size is within a certain definedrange of small particles. However, conventional methods of reducing theparticle size of atovaquone were found to be unsuccessful in producingparticles of the size required to improve bioavailability.

The Microfluidiser has been marketed by the Microfluidics Corporationsince 1985. The principle of its operation is based on a submerged jettechnology. It Has designed, primarily, as a homogenizing device for usein the food and pharmaceutical industries, for the preparation of e.g.emulsion and liposomal systems and has subsequently been used forcell-rupture purposes in biotechnology applications.

It has now surprisingly been found that microfluidised particles ofatovaquone produced using a Microfluidiser have improved bioavailabilityof the compound. It is believed that this is due to the small size andnarrow range of sizes of the microfluidised atovaquone particles.

During operation of the Microfluidiser, the feed stream is pumped into aspecially designed chamber, in which fluid streams interact at very highvelocities and pressures. Fixed microchannels within the interactionchamber provide an extremely focussed interaction zone of intenseturbulence, causing the release of energy amid cavitation and shearforces. Without wishing to be bound by theory it is believed that sinceall product passes through a dimensionally fixed area of energy release,greater size uniformity and smaller sizes are achieved by using theMicrofluidiser rather than conventional methods for producing fineparticles.

Thus, in a first aspect, the present invention provides small particlesof atovaquone. Preferably the particles are microfluidised particles.Suitably at least 90% of the particles have a volume diameter in therange of 0.1-3 μm. Preferably at least 95% of the particles have avolume diameter in the range 0.1-2 μm.

In a second aspect, the present invention provides a pharmaceuticalcomposition comprising particles of atovaquone and one or morepharmaceutically acceptable carriers therefor wherein at least 95% ofthe particles have a volume diameter in the range of 0.1-2 μm.Preferably the particles are microfluidised particles.

The carriers must be acceptable in the sense of being compatible withthe other ingredients of the formula and not deleterious to therecipient thereof

According to a third aspect, the present invention provides a method forthe preparation of microfluidised particles of atovaquone whichcomprises mixing atovaquone with a liquid vehicle to provide a mixturewherein the concentration of atovaquone is less than 450 mg/mL andsubjecting said mixture to at least 3 passes through a Microfluidiser inorder to provide the atovaquone in the form of particles wherein atleast 90% of the particles have a volume diameter in the range 0.1-3 μm.Preferably at least 95% of the particles have a volume diameter in therange 0-1-2 μm.

In a further aspect the present invention provides a method for thepreparation of a pharmaceutical composition comprising the steps of:

a) mixing atovaquone with a liquid vehicle to provide a mixture whereinthe concentration of atovaquone is less than 450 mg/mL.

b) subjecting the mixture to at least 3 passes through a Microfluidiserto provide a microfluidised preparation wherein the atovaquone is in theform of particles and at least 95% of those particles have a volumediameter in the range 0.1-2 μm.

c) mixing the microfluidised preparation with one or morepharmaceutically acceptable carriers therefor.

Suitably, the mixture is subjected to 10-50 passes through theMicrofluidiser, e.g. 25-30 passes. Preferably the mixture is subjectedto 15-25 passes through the Microfluidiser.

In one embodiment, the liquid vehicle is a surfactant. Preferably, theliquid vehicle is a surfactant solution. In a particularly preferredembodiment the surfactant is Poloxamer 188 solution. In anotherpreferred embodiment the pharmaceutically acceptable carriers include asuspending agent. Suitable suspending agents include methyl celluloseand xanthan gum. Preferably the suspending agent is xanthan gum.

Pharmaceutical formulations include those suitable for oral andparenteral (including subcutaneous, intradermal, intramuscular andintravenous) administration as well as administration by naso-gastrictube. Suitable formulations within the scope of the present inventioninclude, for example, solid dosage forms such as tablets and liquiddosage forms, such as suspensions, which are preferred formulations. Theformulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared from the microfluidisedparticles using methods known in the art of pharmacy.

Tests to measure the bioavailability of atovaquone in vivo indicate thatformulations of microfluidised atovaquone have improved bioavailabilitycompared to prior art formulations. The invention therefore provides, ina further aspect, formulations comprising microfluidised atovaquone foruse in therapy, in particular in the treatment and prophylaxis ofprotozoal parasitic infections, e.g. malaria and toxoplasmosis, andinfections caused by P.carinii.

The invention will now be further illustrated by the followingnon-limiting examples:

EXAMPLE 1 Preparation of Microfluidised Particles of Atovaquone

Atovaquone was prepared by methods according to the prior art, e.g. U.S.Pat. No. 5,053,432 (incorporated herein by reference). 600 mL of amixture consisting of 2.5% w/v atovaquone in 0.25% w/v aqueous CelacolM2500 was prepared and 100 mL were retained in a glass jar as a control.A laboratory scale model 120B Microfluidiser was connected to a 90 psipneumatic supply and adjusted to produce a fluid pressure of 15000 psi.The machine base, interaction chamber and pipework of the Microfluidiserwere immersed in a bath of cold water. 500 mL of the mixture were loadedinto the Microfluidiser's bulk vessel and passed through theMicrofluidiser interaction chamber before being returned to the top, andside, of the bulk chamber. The mixture was recirculated continuouslythrough the interaction chamber, and samples were taken at 10, 20, 30,45 and 60 minutes. The number of passes to which each of these sampleshad been subjected was calculated and is shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                               Microfluidisation                                                             time        Sample Volume                                              Sample (minutes)   (ml)          Number of passes                             ______________________________________                                        Control                                                                               0          100           0                                            1      10          105           8                                            2      20          105           9-19                                         3      30          110           31-35                                        4      45          105           65-77                                        5      60           35           142-244                                      ______________________________________                                    

Microscopic observations of the control and samples at 40× magnificationwere made and the results were as follows:

Control--Varied shapes, plates, rods and spheroids, around 5×5 μmgenerally and up to 7.5×10 μm, loosely aggregated.

Sample 1--More rounded smaller shapes, some "large" crystals, lots ofsmall agents 2.5×2.5 μm, more dispersed.

Sample 2--More rounded, smaller shapes, more fragments.

Sample 3--Still more rounded, smaller shapes, more fragments.

Sample 4--Yet more rounder, smaller shapes, more fragments.

Sample 5--Very small particles, all under 2.5 μm all rounded,monodisperse.

EXAMPLE 2 Pharmaceutical Formulation

An oral suspension formulation was prepared by mixing the followingingredients:

    ______________________________________                                        Microfluidised particles of atovaquone                                                              150.0     mg                                            Poloxamer 188         5.0       mg                                            Benzyl alcohol        10.0      mg                                            Xanthan gum           7.5       mg                                            Purified water        to make 1.0                                                                             mL                                            ______________________________________                                    

EXAMPLE 3 Biological Test

Nine healthy fasted male volunteers received single doses of 5 mg/mLsuspensions containing 250 mg atovaquone as a 3 μm mean particle sizesuspension and 1 μm Microfluidised suspension in a randomised crossoverstudy. Plasma samples were taken at intervals up to two weeks after thelast dose and assayed by HPLC. The results are given in table 2 below:

                  TABLE 2                                                         ______________________________________                                                     3 μm suspension                                                                          1 μm suspension                                 ______________________________________                                        mean(SD)AUC  95 (62)μg/mL.h                                                                           247(85)μg/mL.h                                  mean(SD)C    1.2(0.7)μg/mL                                                                            5.0(1.6)μg/mL                                   max                                                                           median T     5 hours       1 hour                                             max                                                                           ______________________________________                                    

The mean (95% CI) increase for the AUC of the 1 μm suspension relativeto the 3 μm suspension was 2.6-fold (1.9-3.5) and for C_(max) was4.1-fold (2.5-6.6).

I claim:
 1. A method for the preparation of microfluidized particles ofatovaquone which comprises mixing atovaquone with a liquid vehicle toprovide a mixture wherein the concentration of atovaquone is less than450 mg/mL and subjecting said mixture to at least 3 passes through aMicrofluidizer.
 2. A method for the preparation of microfluidizedparticles of atovaquone according to claim 1 wherein the microfluldizedparticles have a volume diameter in the range of 0.1-3 μm.
 3. A methodfor the preparation of a pharmaceutical composition which methodcomprises the steps:(a) mixing atovaquone with a liquid vehicle toprovide a mixture wherein the concentration of atovaquone is less than450 mg/mL (b) subjecting the mixture to at least 3 passes through aMicrofluidizer to provide a microfluidized preparation wherein theatovaquone is in the form of particles and at least 90% of thoseparticles have a volume diameter in the range of 0.1-3 μm, (c) mixingthe microfluidized preparation with one or more pharmaceuticallyacceptable carriers therefor.
 4. A method according to claim 2 or claim3 wherein the mixture is subjected to 10 to 50 passes through theMicrofluidizer.
 5. A method according to claim 4, wherein the mixture issubjected to 15-25 passes through the Microfluidizer.
 6. A methodaccording to claim 2 or claim 3 wherein the liquid vehicle is asurfactant solution.
 7. A method according to claim 3, wherein thepharmaceutically acceptable carriers include a suspending agent.
 8. Amethod according to claim 7, wherein the suspending agent is xanthangum.